Luminescence method has been used as an optical method to quantify the number of analyte or molecules. Luminescence reactions including light-emitting chemical reactions (chemiluminescence, CL), light-emitting biological reactions (bioluminescence, BL), and electro-induced luminescence have a diverse range of analytical and biological applications. Advantages of luminescence assays include very high sensitivity due to the current technology in photon counting and enzyme amplification, rapid signal generated in a few seconds, and assays do not need an external excitation light source. In many situations, these procedures are replacing the use of radioactive nuclides. As luminescent agents become more efficient, many more studies are making use of luminescence assays as analytical tools. Chemiluminescent substrates, such as dioxetane, luminol, acridinium ester and hydrazide, have been developed. These compounds are catalyzed by hydrolytic enzymes and the resulting products emit light. Bioluminescent reactions are generally more efficient than chemiluminescence. BL has traditionally been associated with firefly luciferase. AquaLite (SeaLite Sciences, Georgia) is a recombinant form of a photoprotein from jellyfish. It can be triggered to produce all of its light in a single step within a few seconds. CL and BL methods have been developed for many enzyme labels (alkaline phosphatase, galactosidase, horseradish peroxidase, etc.). The enzymes are conjugated to the secondary antibody or analyte for subsequent substrate reactions.
In a typical sandwich immunoassay, the analyte is sandwiched between the antibody conjugate and immobilized probes. The luminescence intensity at any time is a direct measure of the concentration of enzyme conjugate or analyte for positive identification. The newly developed dioxetane offers a detection sensitivity of 600 molecules (10−21 mole), making it several orders more sensitive than the fluorescence-based assay. Rather than a luminescent species being directly attached to a target analyte or to its binding partner, an enzyme is used to catalyze a luminescent reaction. The catalytic turnover ability of the enzyme allows thousands of potentially luminescent reactions to occur per second as long as sufficient substrate is present. Less than 10−21 mole of alkaline phosphatase can be detected in solutions using dioxetane-based compounds, such as Lumi-PPD and Lumi-PS-1 of Lumigen Inc. (Southfield, Mich.). When Lumi-PPD is added to a microwell containing alkaline phosphatase, the resulting chemiluminescence reaches a maximum after 5-10 minutes and remains constant for more than an hour. Various luminescence detection devices (for example, a luminometer) are commercially available.
As technology advances, it becomes necessary to detect very small amount of analyte in femtomole, attomole, zetomole, or single molecule quantity. For high sensitivity optical detection, photon multiplier tube (PMT) is the most widely used detector. The PMT has been operated in two different modes: photocurrent and photoncounting. The photocurrent mode is designed to detect high light signal with abundance of optical photons. If the input photons are continuously coming into a PMT, it will be a photocurrent application. However, if the number of input photons is low, for example, each photo is coming into PMT apart from each other, the photocurrent measurement will fail. It is because that there is no analog to digital converter (ADC) which can perform conversion fast enough to be able to measure the photocurrent produced by one photon.
The number of photons being generated in the reaction is directly proportional to the analyte quantity. Optical detection with photoncounting capability becomes critical for the studies of events that occur with a very small amount of analyte. Photoncounting is one of the best methods to detect molecular events. The existing photoncounting system is based on the combination of a PMT and a photoncounting circuitry. Photoncounting PMT has the ability to count photons and detect a single photon event, such as photons generated from luminescence or single molecular fluorescence. While the photoncounting PMT has the sensitivity to detect every single photon and count the number of photons, it has drawbacks of optical nonlinearity and inaccuracy. The photons reaching the detector have not been counted correctly. Typically, a PMT outputs an electrical pulse in correspondence to the entering photon. One photon generates one pulse. However, when the input light signal is slightly increased, the photons may overlap and cause the electrical pulse to overlap accordingly. The overlapped pulses become a long pulse, rather than individual short pulses. Since photoncounting circuitry measures light intensity by counting the number of the electrical pulses, the long pulse will be considered as one pulse. In this situation, the photoncounting system is not able to count the photons accurately and the optical nonlinearity would occur.
U.S. Pat. No. 5,401,951 to Butler et al., entire contents of which are incorporated herein by reference, discloses an method and apparatus for overload protection for a photomultiplier tube. A light source illuminates a photomultiplier tube which produces a signal proportional to the incoming radiation which is sent to photon counting electronics. The photon counting electronics produces a signal in proportion to the input photons to the photomultiplier tube and also provides an output to a frequency to voltage converter. The frequency to voltage converter is used to modulate a high voltage amplifier which controls the output of the photomultiplier tube. When the photon counting electronics indicate to the frequency to voltage converter that the photons produced by the photomultiplier tube exceed a predetermined maximum the high voltage amplifier reduces the gain of the photomultiplier tube.
U.S. Pat. No. 6,596,980 to Rusu et al., entire contents of which are incorporated herein by reference, discloses a method and apparatus for overload protection for a photomultiplier tube. A method and apparatus to measure statistical variation of electrical signal phase in integrated circuits using time-correlated photon counting
U.S. Pat. No. 6,342,701 to Kash et al., entire contents of which are incorporated herein by reference, discloses a system for time-correlated photon counting. The system uses one or more photon detectors to produce electrical pulses corresponding to photons read from a target. The system uses a discriminator with a first input coupled to a trigger output from a pulsed optical source and a second input for receiving the electrical pulses.
None of the above-identified patents discloses a method to count the number of single photons. The invention includes apparatus and methods to accurately count the individual photons using a timing counter and intensity discriminator for measuring the overlapped pulses.